MicroRNAs (miRNAs) are an abundant class of small (~22 nt) regulatory RNAs that control the expression of more than half of all protein-coding genes in humans and are essential components in diverse mammalian physiological processes. Many miRNAs contribute to cellular homeostasis and aberrant miRNA expression is often associated with dysregulated cell growth and cancer. Despite the widespread function of miRNAs in human biology there are fundamental gaps in understanding the molecular mechanisms ensuring fidelity in miRNA biogenesis. Continued existence of these gaps represents a significant medical problem because, until it is filled, understanding of genetic abnormalities in miRNA biogenesis factors that promote tumor growth will remain largely incomprehensible. The long-term goal is to understand the miRNA pathway and related RNA silencing processes to provide detailed insights that can be harnessed for the diagnosis and treatment of human disease. The objective of this application is to dissect the mechanism of the human RISC-loading complex (RLC), which is a large molecular assembly that catalyzes the final steps of miRNA biogenesis and determines which cellular genes are to be down-regulated by the miRNA pathway. The proposed work will explore the hypothesis that genetic mutations in RLC components associated with cancer act by perturbing the mechanism of miRNA biogenesis, leading to aberrant miRNA expression, which in turn drives tumorigenesis. The rational for the proposed research is that detailed understanding of the structure and mechanism of the RLC will provide molecular insights into the biology of diverse human tumors. Guided by strong preliminary data, this objective will be achieved by pursuing two specific aims: 1) Determine structures of the RISC-loading Complex; and 2) Dissect the mechanisms of RISC-loading and guide RNA selection. Under the first aim, X-ray crystallography and electron microscopy will be used to determine molecular structures of the human RLC and its individual components. These efforts will be facilitated by innovative methods for examining small particles by electron microscopy and crystals that are already in hand. Completed results will significantly advance the RNA silencing field because, currently, the only published RLC structure has been shown to be incorrect and the details of how the complex assembles have become muddled and contested in the literature. Under the second aim, discrete RISC-loading steps in wild-type and mutant RLCs will be examined using established biochemical assays. This aim is significant because it will provide mechanistic details into the process of RISC- loading, which is currently only understood at an empirical level, as well as direct and specific insights into how mutations the drive tumorigenesis perturb the mechanisms in miRNA biogenesis. These results will thereby inform efforts to understand and treat diverse forms of cancer. The overall proposal is innovative because it identifies and addresses a specific clinical exigency for biological insight that can be mitigated through proven structural and biochemical approaches.